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Columnar apple primary roots share some features of the columnar-specific gene expression profile of aerial plant parts as evidenced by RNA-Seq analysis.

Petersen R, Djozgic H, Rieger B, Rapp S, Schmidt ER - BMC Plant Biol. (2015)

Bottom Line: Radicles mainly express genes associated with primary metabolism, growth and development.Homozygous columnar embryos are viable and form normal radicles under natural conditions, and selection towards heterozygous plants most likely occurs due to breeders' preferences.Cell wall and phytohormone biosynthesis and metabolism experience differential regulation in columnar radicles.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Johannes Gutenberg-University, Mainz, D-55128, Germany. peterser@uni-mainz.de.

ABSTRACT

Background: Primary roots (radicles) represent the first visible developmental stages of the plant and are crucial for nutrient supply and the integration of environmental signals. Few studies have analyzed primary roots at a molecular level, and were mostly limited to Arabidopsis. Here we study the primary root transcriptomes of standard type, heterozygous columnar and homozygous columnar apple (Malus x domestica) by RNA-Seq and quantitative real-time PCR. The columnar growth habit is characterized by a stunted main axis and the development of short fruit spurs instead of long lateral branches. This compact growth possesses economic potential because it allows high density planting and mechanical harvesting of the trees. Its molecular basis has been identified as a nested Gypsy-44 retrotransposon insertion; however the link between the insertion and the phenotype as well as the timing of the phenotype emergence are as yet unclear. We extend the transcriptomic studies of columnar tissues to the radicles, which are the earliest developmental stage and investigate whether homozygous columnar seedlings are viable.

Results: Radicles mainly express genes associated with primary metabolism, growth and development. About 200 genes show differential regulation in a comparison of heterozygous columnar radicles with non-columnar radicles, whereas the comparison of homozygous columnar radicles with non-columnar radicles yields about 300 differentially regulated genes. Genes involved in cellulose and phenylpropanoid biosynthesis, cell wall modification, transcription and translation, ethylene and jasmonate biosynthesis are upregulated in columnar radicles. Genes in the vicinity of the columnar-specific Gypsy-44 insertion experience an especially strong differential regulation: the direct downstream neighbor, dmr6-like, is downregulated in heterozygous columnar radicles, but strongly upregulated in columnar shoot apical meristems.

Conclusions: The transcriptomic profile of primary roots reflects their pivotal role in growth and development. Homozygous columnar embryos are viable and form normal radicles under natural conditions, and selection towards heterozygous plants most likely occurs due to breeders' preferences. Cell wall and phytohormone biosynthesis and metabolism experience differential regulation in columnar radicles. Presumably the first step of the differential regulation most likely happens within the region of the retrotransposon insertion and its tissue-specificity suggests involvement of one (or several) tissue-specific regulator(s).

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Differential gene expression in theCotarget region. Fold changes of RNA-Seq samples (upper panel) and n-fold expression of qRT-PCR experiments (lower panel) in different tissues were calculated for up to eight genes located in the vicinity of Gypsy-44 as well as for Gypsy-44 itself in at least two biological replicates. Positions on the metacontig are given. Blue bars indicate fold changes for the non-columnar sample (normalized to 1), red and light pink bars indicate fold changes in the heterozygous and homozygous columnar sample, respectively, for a comparison with the non-columnar sample. Two parallel lines on a bar and the y axis represent a broken axis. Absolute read counts of 0 or 1 in both genotypes of a tissue are considered no expression (n.e.). Error bars in qRT-PCR bar chart represent standard deviations of three technical replicates. The two orange boxes below the two panels of graphs signify zoom-ins on the dmr6-like graphs within the region 0 – 2.
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Fig5: Differential gene expression in theCotarget region. Fold changes of RNA-Seq samples (upper panel) and n-fold expression of qRT-PCR experiments (lower panel) in different tissues were calculated for up to eight genes located in the vicinity of Gypsy-44 as well as for Gypsy-44 itself in at least two biological replicates. Positions on the metacontig are given. Blue bars indicate fold changes for the non-columnar sample (normalized to 1), red and light pink bars indicate fold changes in the heterozygous and homozygous columnar sample, respectively, for a comparison with the non-columnar sample. Two parallel lines on a bar and the y axis represent a broken axis. Absolute read counts of 0 or 1 in both genotypes of a tissue are considered no expression (n.e.). Error bars in qRT-PCR bar chart represent standard deviations of three technical replicates. The two orange boxes below the two panels of graphs signify zoom-ins on the dmr6-like graphs within the region 0 – 2.

Mentions: In order to identify the link between the columnar-specific Gypsy-44 insertion and the columnar growth habit (most likely corresponding to the first, causal level of differential gene expression) we performed detailed expression analyses of all genes in the vicinity of the Gypsy-44 insertion. Eight genes that had previously been found to be transcribed and were annotated on our BAC metacontig based on the transcriptomic data [10] as well as Gypsy-44 itself were used for in-depth RNA-Seq and qRT-PCR analyses (Table 5, Figure 5). Fold changes were calculated for the primary root datasets as well as for three RNA-Seq Illumina datasets each, generated from shoot apical meristems of A14 and P28 ([13,14], [EMBL: PRJEB2506]) and two transcriptomic datasets each, obtained from the total RNA of a McIntosh and a Wijcik leaf ([10], [EMBL: PRJEB1902]). If all genotypes of one tissue displayed read counts of 0 or 1 for a specific gene, this gene was considered not to be expressed (numeric read counts and fold changes can be found in Additional file 7). The results are shown in the upper part of Figure 5. At1g08530-like and MDP0000934866 (At1g06150-like) showed similar expression levels for columnar and non-columnar varieties in all samples analyzed. For MDP0000927091 (Autophagy9-like) and 5NG4-like, similar levels of transcription were reached in all but the SAM3 dataset, in which they were induced in the columnar sample when compared with the non-columnar sample. Fold changes were less consistent between different tissues and across biological replicates for MDP0000912172 (PP2C15-like), MDP0000163720 (ACC1-like) and Gypsy-44 itself. For the former two genes, this might be caused by the generally lower expression level making fold changes prone to fluctuation. The most striking results were obtained for dmr6-like and MDP0000927098 (ATL5K-like), the first two protein coding genes that follow downstream of the Gypsy-44 insertion: they were downregulated in the primary root samples and strongly upregulated in the shoot apical meristem of columnar varieties. These effects were most pronounced for dmr6-like in the shoot apical meristem, where no or only basal transcription (0 reads or 1 read) occurred in non-columnar A14, while expression was 30-, 53- and 56-times higher in the three biological replicates of P28.Table 5


Columnar apple primary roots share some features of the columnar-specific gene expression profile of aerial plant parts as evidenced by RNA-Seq analysis.

Petersen R, Djozgic H, Rieger B, Rapp S, Schmidt ER - BMC Plant Biol. (2015)

Differential gene expression in theCotarget region. Fold changes of RNA-Seq samples (upper panel) and n-fold expression of qRT-PCR experiments (lower panel) in different tissues were calculated for up to eight genes located in the vicinity of Gypsy-44 as well as for Gypsy-44 itself in at least two biological replicates. Positions on the metacontig are given. Blue bars indicate fold changes for the non-columnar sample (normalized to 1), red and light pink bars indicate fold changes in the heterozygous and homozygous columnar sample, respectively, for a comparison with the non-columnar sample. Two parallel lines on a bar and the y axis represent a broken axis. Absolute read counts of 0 or 1 in both genotypes of a tissue are considered no expression (n.e.). Error bars in qRT-PCR bar chart represent standard deviations of three technical replicates. The two orange boxes below the two panels of graphs signify zoom-ins on the dmr6-like graphs within the region 0 – 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352258&req=5

Fig5: Differential gene expression in theCotarget region. Fold changes of RNA-Seq samples (upper panel) and n-fold expression of qRT-PCR experiments (lower panel) in different tissues were calculated for up to eight genes located in the vicinity of Gypsy-44 as well as for Gypsy-44 itself in at least two biological replicates. Positions on the metacontig are given. Blue bars indicate fold changes for the non-columnar sample (normalized to 1), red and light pink bars indicate fold changes in the heterozygous and homozygous columnar sample, respectively, for a comparison with the non-columnar sample. Two parallel lines on a bar and the y axis represent a broken axis. Absolute read counts of 0 or 1 in both genotypes of a tissue are considered no expression (n.e.). Error bars in qRT-PCR bar chart represent standard deviations of three technical replicates. The two orange boxes below the two panels of graphs signify zoom-ins on the dmr6-like graphs within the region 0 – 2.
Mentions: In order to identify the link between the columnar-specific Gypsy-44 insertion and the columnar growth habit (most likely corresponding to the first, causal level of differential gene expression) we performed detailed expression analyses of all genes in the vicinity of the Gypsy-44 insertion. Eight genes that had previously been found to be transcribed and were annotated on our BAC metacontig based on the transcriptomic data [10] as well as Gypsy-44 itself were used for in-depth RNA-Seq and qRT-PCR analyses (Table 5, Figure 5). Fold changes were calculated for the primary root datasets as well as for three RNA-Seq Illumina datasets each, generated from shoot apical meristems of A14 and P28 ([13,14], [EMBL: PRJEB2506]) and two transcriptomic datasets each, obtained from the total RNA of a McIntosh and a Wijcik leaf ([10], [EMBL: PRJEB1902]). If all genotypes of one tissue displayed read counts of 0 or 1 for a specific gene, this gene was considered not to be expressed (numeric read counts and fold changes can be found in Additional file 7). The results are shown in the upper part of Figure 5. At1g08530-like and MDP0000934866 (At1g06150-like) showed similar expression levels for columnar and non-columnar varieties in all samples analyzed. For MDP0000927091 (Autophagy9-like) and 5NG4-like, similar levels of transcription were reached in all but the SAM3 dataset, in which they were induced in the columnar sample when compared with the non-columnar sample. Fold changes were less consistent between different tissues and across biological replicates for MDP0000912172 (PP2C15-like), MDP0000163720 (ACC1-like) and Gypsy-44 itself. For the former two genes, this might be caused by the generally lower expression level making fold changes prone to fluctuation. The most striking results were obtained for dmr6-like and MDP0000927098 (ATL5K-like), the first two protein coding genes that follow downstream of the Gypsy-44 insertion: they were downregulated in the primary root samples and strongly upregulated in the shoot apical meristem of columnar varieties. These effects were most pronounced for dmr6-like in the shoot apical meristem, where no or only basal transcription (0 reads or 1 read) occurred in non-columnar A14, while expression was 30-, 53- and 56-times higher in the three biological replicates of P28.Table 5

Bottom Line: Radicles mainly express genes associated with primary metabolism, growth and development.Homozygous columnar embryos are viable and form normal radicles under natural conditions, and selection towards heterozygous plants most likely occurs due to breeders' preferences.Cell wall and phytohormone biosynthesis and metabolism experience differential regulation in columnar radicles.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Johannes Gutenberg-University, Mainz, D-55128, Germany. peterser@uni-mainz.de.

ABSTRACT

Background: Primary roots (radicles) represent the first visible developmental stages of the plant and are crucial for nutrient supply and the integration of environmental signals. Few studies have analyzed primary roots at a molecular level, and were mostly limited to Arabidopsis. Here we study the primary root transcriptomes of standard type, heterozygous columnar and homozygous columnar apple (Malus x domestica) by RNA-Seq and quantitative real-time PCR. The columnar growth habit is characterized by a stunted main axis and the development of short fruit spurs instead of long lateral branches. This compact growth possesses economic potential because it allows high density planting and mechanical harvesting of the trees. Its molecular basis has been identified as a nested Gypsy-44 retrotransposon insertion; however the link between the insertion and the phenotype as well as the timing of the phenotype emergence are as yet unclear. We extend the transcriptomic studies of columnar tissues to the radicles, which are the earliest developmental stage and investigate whether homozygous columnar seedlings are viable.

Results: Radicles mainly express genes associated with primary metabolism, growth and development. About 200 genes show differential regulation in a comparison of heterozygous columnar radicles with non-columnar radicles, whereas the comparison of homozygous columnar radicles with non-columnar radicles yields about 300 differentially regulated genes. Genes involved in cellulose and phenylpropanoid biosynthesis, cell wall modification, transcription and translation, ethylene and jasmonate biosynthesis are upregulated in columnar radicles. Genes in the vicinity of the columnar-specific Gypsy-44 insertion experience an especially strong differential regulation: the direct downstream neighbor, dmr6-like, is downregulated in heterozygous columnar radicles, but strongly upregulated in columnar shoot apical meristems.

Conclusions: The transcriptomic profile of primary roots reflects their pivotal role in growth and development. Homozygous columnar embryos are viable and form normal radicles under natural conditions, and selection towards heterozygous plants most likely occurs due to breeders' preferences. Cell wall and phytohormone biosynthesis and metabolism experience differential regulation in columnar radicles. Presumably the first step of the differential regulation most likely happens within the region of the retrotransposon insertion and its tissue-specificity suggests involvement of one (or several) tissue-specific regulator(s).

Show MeSH